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Abstract Conventional methods for sampling hormones often preclude strong inference experimental designs, including repeated measures of both hormones and behavior and balanced or simultaneous designs for hormone-behavior sampling. In amphibians there is an opportunity to non-invasively and repeatedly sample excreted steroids in the water. We examined testosterone (T) in túngara frogs (Physalaemus (=Engystomops) pustulosus) using minimally invasive water-borne methods. First, we validated procedures for the collection, extraction and measurement of T in adult males and females using pharmacological challenges coupled with estimates of parallelism and recovery determination. Next, we extended the timeline of sampling over 9 days in order to evaluate the kinetics of excretion (baseline phase, challenge phase, recovery phase), including the estimation of individual differences during baseline sampling. We also estimated concentrations of creatinine (Cr) in each water sample and evaluated whether correcting for this proxy of urine concentration significantly decreased error variance in T estimates. Lastly, we incorporated a standardized and repeated measures assay of male sexual proceptivity (phonotaxis) during the predicted peak T and recovery T timepoints. We found strong evidence supporting the utility of these methods for precise, biologically informative estimates of T in both sexes. Males had higher T than females and responded to pharmacological challenges by elevating T substantially within 48 h of challenge (hCG, GnRH). Males exhibited repeatability in baseline T and phonotaxis frequencies were positively associated with higher T. Adjusting T levels for the simultaneous measure of Cr significantly improved model fit, which in conjunction with marked variation in urine concentration, suggests that urine likely serves as the major source of excreted T. In summary, this proof-of-concept and methods study demonstrates the utility and accuracy of measuring water-borne T and behavior in amphibians.